EP2524221B1 - Systeme zum zählen von zellen und biomolekülen - Google Patents

Systeme zum zählen von zellen und biomolekülen Download PDF

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Publication number
EP2524221B1
EP2524221B1 EP11733248.6A EP11733248A EP2524221B1 EP 2524221 B1 EP2524221 B1 EP 2524221B1 EP 11733248 A EP11733248 A EP 11733248A EP 2524221 B1 EP2524221 B1 EP 2524221B1
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Prior art keywords
sample
fluorescent
bright
light
cells
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English (en)
French (fr)
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EP2524221A2 (de
EP2524221A4 (de
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Leo L. Chan
Peter Li
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Nexcelom Bioscience LLC
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Nexcelom Bioscience LLC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G01N15/1433—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/10—Arrangements of light sources specially adapted for spectrometry or colorimetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J5/00—Radiation pyrometry, e.g. infrared or optical thermometry
    • G01J5/10—Radiation pyrometry, e.g. infrared or optical thermometry using electric radiation detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1486—Counting the particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N2021/1734—Sequential different kinds of measurements; Combining two or more methods
    • G01N2021/1736—Sequential different kinds of measurements; Combining two or more methods with two or more light sources
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N2021/1738—Optionally different kinds of measurements; Method being valid for different kinds of measurement
    • G01N2021/174—Optionally different kinds of measurements; Method being valid for different kinds of measurement either absorption-reflection or emission-fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6491—Measuring fluorescence and transmission; Correcting inner filter effect
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3581—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/062—LED's
    • G01N2201/0627—Use of several LED's for spectral resolution

Definitions

  • the invention generally relates to analytical and monitoring systems useful for analyzing and measuring cells and biological samples. More particularly, the invention relates to systems for counting cells and biological molecules in solution samples.
  • An important aspect in the fields of medical diagnostics and biomedical research involves detection, identification, quantification, and characterization of various cells and biomolecules of interest through testing of biological samples such as blood, spinal fluid, cell culture and urine. Healthcare providers and biomedical researchers routinely analyze such biological samples for the microscopic presence and concentrations of cells and biomolecules.
  • Fluorescence microscopy, fluorescent plate reader, and flow cytometry are traditionally used for fluorescence detection with cell-based assays. These methods utilize glass slides, microtiter plates, and flow chamber to perform fluorescence analysis. These fluorescence detection methods, however, often incorporate expensive excitation light sources such as lasers or arc lamps for high intensity excitation. Typically, there is an excitation light source and a detection probe with an emission filter to pick up specific fluorescent signals. In an instrument such as fluorescent microscope, for example, a dichroic filter is required to reflect the light from the top normal (i.e., at a right angle) to the sample chamber. The emitted fluorescence is then picked up by passing through the dichroic filter into a detector such as a camera, spectrometer, etc. These fluorescence detection methods are also not used to directly measure sample concentration, since they do not measure samples in a specific fluid volume.
  • An example of a previous fluorescent cell counting technology is one that utilizes a filter cube (such as those provided by Omega Optical) in an assembly that includes optics, a camera, and a sample holder.
  • a filter cube such as those provided by Omega Optical
  • Such as assembly may provide sufficient fluorescent images of cells and other biosamples for a number of applications, and it provides a simple and efficient method to generate fluorescent images of biological samples.
  • This technology utilizes a fixed chamber volume, which is used to directly measure sample concentration, while analyzing fluorescence intensity. It, however, lacks the sensitivity for low fluorescent signal detection and imaging.
  • One issue with such a system for example, is that the excitation light may leak out into the emission filter in the filter cube.
  • the filter cube format generally is inflexible in color selection. Only one specific filter cube and one LED could be used for one color. There is not much space in the instrument to incorporate other colors, making it difficult to allow multi-color applications.
  • the invention is based, in part, on the discovery of a unique design approach resulting in a much improved system for detecting and imaging (e.g., measuring, analyzing, counting, or monitoring) microscopic objects.
  • the system includes an unconventional design for fluorescent excitation such that the system allows two or multiple oblique incident excitation beams. This novel approach significantly liberates the overall design of the system and enables multiple light sources to be placed with various incident angles.
  • the invention presented here describes a novel method for maximizing the fluorescent signal of the sample while minimizing the background noise of sample slides through the utilization of oblique excitation light sources and multiple LEDs (light-emitting diodes) illumination.
  • oblique excitation light sources and multiple LEDs light-emitting diodes
  • the new system paves the way for enhanced (i.e., more powerful) excitation by putting in place two or multiple excitation sources, it also enables flexible use of and selection for wavelengths and incident angles of the excitation beams. By allowing different combinations of angles of incident excitation beams, images may be generated that were not possible or could not be easily generated using prior systems.
  • the invention generally relates to a system as defined in appended independent claim 1. Particular embodiments are defined in the appended dependent claims.
  • the at least two fluorescent light sources are capable of simultaneously providing two excitation light beams having the same (or different) wavelengths to the sample.
  • the fluorescent excitation light beam(s) is at an incident angle of about 30° to about 70°, about 35° to about 55°, about 10° to about 45°, greater than about 45° and less than about 80°, or about 45° in relation to the plane surface of the sample chamber window.
  • the cells or biomolecules that may be counted include, but are not limited to, bacteria, algae, fungi, mammalian cells, insect cells, plant cells, proteins, DNA molecules, and surface markers.
  • the covered sample chamber has a fixed depth, ranging from about 1 ⁇ m to about 200 ⁇ m. In some preferred embodiments, the fixed depth ranges from about 10 ⁇ m to about 100 ⁇ m.
  • the covered sample chamber is configured to hold a sample volume of about 1 ⁇ L to about 1,000 ⁇ L. In certain preferred embodiments, the covered sample chamber is configured to hold a sample volume of about 1 ⁇ L to about 500 ⁇ L (e.g., about 1 ⁇ L to about 100 ⁇ L).
  • the fluorescent light sources emit a beam having a wavelength ranging from about 300 nm to about 10,000 nm (e.g., from about 300 nm to about 2,000 nm or from about 300 nm to about 1,000 nm).
  • a fluorescent image is acquired from excitation by directing at least two excitation light beams to the sample. In certain other preferred embodiments, the fluorescent image is acquired from excitation by directing multiple (e.g., three, four) excitation light beams to the sample.
  • the invention relates to systems for counting cells and biological molecules.
  • the invention is based, in part, on the discovery of a unique design that enables in a much improved system for detecting and imaging (e.g., measuring, analyzing, counting or monitoring) microscopic objects in solution samples.
  • the system includes an unconventional approach to fluorescent excitation such that it allows two or multiple oblique incident excitation beams that are simultaneously operatable.
  • This novel approach significantly revolutionizes and liberates the overall design of an imaging system and enables multiple (same or different) light sources to be placed with various incident angles to the sample.
  • the new system thus paves the way for enhanced excitation power by putting in place two or multiple excitation sources. It additionally allows the flexible use of and selection of wavelengths and incident angles of excitation beams. With different and combinations of angles of incident excitation beams, one can detect and generate images that were not possible or easily generated using prior systems.
  • FIG. 1 Depicted in FIG. 1 is an example of a prior art cell-counting system.
  • the system includes a counting chamber, a fluorescent light source (connected to a fluorescent light beam narrowing device), and a bright-field light source (connected to a bright-field light beam narrowing device).
  • the cell counting system in FIG. 1 further includes a detection device (camera), and a fluorescent filter assembly that allows excitation light from the fluorescent light source to illuminate the sample in the chamber.
  • the fluorescent excitation light shines on the sample in the counting chamber at a right (normal) angle.
  • the present invention significantly improves the sensitivity and detection limit of the above-system, at least in two major aspects.
  • an oblique incidence excitation light source is used to minimize the background signal due to light leakage from excitation filter to emission filter that is common in a filter cube method.
  • the cell counting system 200 includes at least one fluorescent light source 210 (only one shown) connected to a fluorescent light beam narrowing device 215, a bright-field light source 220 connected to a bright-field light beam narrowing device 225.
  • the system of the present invention as defined in appended claim 1, comprises two or more fluorescent light sources 210.
  • the cell counting system further includes a counting chamber 230 and a detection device 240 (camera). In this system, the fluorescent excitation light shines on the sample 250 in the counting chamber 230 at an oblique angle.
  • the cell counting system 200 further includes a microscope objective 206 and a movable light shutter 228.
  • the detection device can be a camera, such as a CCD (charge-coupled device) camera, for acquiring images.
  • the camera may be fitted with a cooling capability.
  • microscope objective movements are under the control of a computer 260 operably connected to the system 200.
  • the counting chamber 230 has a a fixed depth ranging from 1 ⁇ m to 200 ⁇ m and is configured to hold a sample volume of about 1 ⁇ L to 1,000 ⁇ L of a suspension of cells or biomolecules in a sample.
  • the counting chamber 230 is covered such that the suspension of sample therein would not lose volume due to evaporation.
  • the chamber 230 may be loaded with a sample by pipetting the sample into the sample introduction port of the chamber 230. As the sample is loaded into the chamber, air escapes the chamber 230 through the air escape port in the chamber 230.
  • An exemplary sample size is 20 ⁇ l although, within the scope of the appended claim 1, actual sample size may vary according to the application and instrument setup.
  • the chamber 230 is loaded into the counting system 200 through a slot in a housing of the system.
  • the fluorescent light source and the bright-field light source can be a light emitting diode.
  • the fluorescent light beam narrowing device and the bright-field light beam narrowing device can be collimators.
  • the sample volume under interrogation can be obtained from the height of the counting chamber and the area of the sample that is imaged.
  • the interrogated sample volume can be obtained and is known for each image taken. It should be noted that the chamber height may be varied from application to application as long as the interrogated sample volume can be obtained or is known.
  • System 200 is configured for bright-field imaging and fluorescent imaging of the sample in the chamber 230.
  • the components of the cell counting system 200 are encased in a housing.
  • the bright-field light source 220 is positioned at the base of the housing and is configured to emit light onto the sample in the chamber 230 positioned in-line above the bright-field light source 220.
  • a bright-field light beam narrowing device 225 Between the chamber 230 and the bright-field light source 220 is a bright-field light beam narrowing device 225.
  • the beam narrowing device focuses the light emitted from the bright-field light source 220, and directs the light onto the sample in the chamber 230.
  • Also positioned between the chamber 230 and the bright-field light source 220 is a movable light shutter 228.
  • the movable light shutter 228 is located above the bright-field light beam narrowing device 225 and below the chamber 230.
  • the light shutter 228 is connected to a mechanism for moving the shutter, such as a motor or a solenoid.
  • the light shutter 228 is mechanically moved out of line with the bright-field light source 220 to allow the light from the bright-field light source 220 to interact with the sample in the chamber 230 during bright-field imaging.
  • the light shutter 228 is mechanically moved in-line with the bright-field light source 220 to block the light from the bright-field light source 220 from interacting with the sample in the chamber 230 during fluorescent imaging.
  • the microscope objective 206 is responsible for primary image formation and is involved in determining quality of images that the system 200 is capable of producing. Microscope objective 206 is also involved in determining the magnification of a particular sample and the resolution under which fine sample details may be observed in the system 200. Microscope objectives are commercially available from Olympus America Inc. (Center Valley, PA).
  • the detection device 240 After the light from the bright-field light source 220 passes through the microscope objective 206, the emitted light from the sample passes through an emission filter 265, and the emitted light from the sample in the chamber 230 is acquired by the detection device 240.
  • the emission filter 265 is in-line with the bright-field light source 220, the bright-field beam narrowing device 225, the chamber 230, the microscope objective 206, and the detection device 240.
  • An exemplary detection device is a CCD camera commercially available from Olympus America Inc. (Center Valley, PA).
  • the image from the detection device 240 is transmitted to a computer 260 having analysis software 211.
  • the system includes at least one fluorescent light source 210 for fluorescent imaging of the sample in the chamber 230.
  • the system of the present invention as defined in appended claim 1, comprises two or more fluorescent light sources 210.
  • the fluorescent light source 210 emits excitation light through a fluorescent beam narrowing device 215.
  • the light shutter 228 is mechanically moved in-line with the bright-field light source 220 to block the white light from the bright-field light source 220 from interacting with the sample in the chamber 230 during fluorescent imaging.
  • FIG. 2 shows one set of fluorescent light source and fluorescent beam narrowing device.
  • two or multiple sets of fluorescent light sources and fluorescent beam narrowing devices are used for fluorescence excitation and emission detection.
  • FIG. 3 illustrates such a system with two sets of fluorescent excitation light sources. With two or multiple (e.g., 3 or 4) sets of fluorescence excitation and emission available on the same sample, variable and/or stronger excitations may be achieved, as well as more than one fluorescent label may be used for advanced assays.
  • FIGs. 4A and 4B are pictures showing different views of an exemplary cell counting system.
  • the cell counting system of the invention includes an unconventional design for fluorescent excitation such that the system allows two or multiple oblique incident excitation beams.
  • This novel approach significantly liberates the overall design of the system and enables multiple light sources to be placed with various incident angles.
  • the cell counting system described herein captures bright-field and fluorescent images of cells or biomolecules in the chamber, analyzes the number of cells or biomolecules, sizes and fluorescent intensity of each cell, and then converts this data to concentration, size and fluorescence histograms and scatter plots.
  • the cell counting system of the invention is useful for various biological assays and other applications.
  • the invention generally relates to a system for counting cells or biomolecules, as defined in appended claim 1.
  • the at least two fluorescent light sources are capable of simultaneously providing two excitation light beams having the same (or different) wavelengths to the sample.
  • the fluorescent excitation light beam(s) is (are) at an incident angle of about 30° to about 70°, about 35° to about 55°, about 10° to about 45°, greater than about 45° and less than about 80°, or about 45° in relation to the plane surface of the sample chamber window.
  • the two or multiple fluorescent excitation light beams have the same incident angle in relation to the window's plane surface. In certain embodiments, the two or multiple fluorescent excitation light beams have a combination of different incident angles in relation to the window's plane surface.
  • the two or multiple fluorescent excitation light beams have different wavelengths. In certain embodiments, the two or multiple fluorescent excitation light beams have the same wavelengths.
  • the excitation light sources may have selectable excitation wavelengths.
  • the cells or biomolecules that are counted include, but are not limited to, bacteria, algae, fungi, mammalian cells, insect cells, plant cells, proteins, DNA molecules, and surface markers.
  • the sample chamber is covered.
  • the sample chamber has a fixed depth, ranging from 1 ⁇ m to about 200 ⁇ m (e.g., from about 10 ⁇ m to about 100 ⁇ m).
  • the covered sample chamber is configured to hold a sample volume of about 1 ⁇ L to about 1,000 ⁇ L. In certain preferred embodiments, the covered sample chamber is configured to hold a sample volume of about 1 ⁇ L to about 500 ⁇ L (e.g., about 1 ⁇ L to about 100 ⁇ L).
  • the fluorescent light sources emit a beam having a wavelength ranging from about 300 nm to about 10,000 nm (e.g., from about 300 nm to about 2,000 nm or from about 300 nm to about 1,000 nm).
  • a fluorescent image is acquired from excitation by directing at least two excitation light beams to the sample.
  • the fluorescent image is acquired from excitation by directing multiple (e.g., three, four) excitation light beams to the sample.
  • Each of the excitation light beams may have the same (or different) oblique angle to the bright-field light beam.
  • the excitation light beams are at about 45° angle to the bright-field light beam and at an incident angle of 10° to 80° to the window's plane surface.
  • the excitation light beams are less than about 45° angle to the bright-field light beam and at an incident angle of 10° to 80° to the window's plane surface. In certain embodiments, the excitation light beams are greater than about 45° angle to the bright-field light beam and at an incident angle of 10° to 80° to the window's plane surface.
  • the invention has at least two unique advantages over the previous design.
  • the first advantage is the flexibility for multi-emission incorporation, which utilizes multiple fluorescent light sources, such as LEDs, and multiple filters to perform multi-color fluorescence detection.
  • the second unique advantage is the reduction of background caused the light leaking from excitation to the emission filter. Furthermore, since the excitation light is at an oblique incidence, less scattered light reflects back through the objective, thereby further reducing the background.
  • Samples that may be analyzed using the systems of the invention include cells or biomolecules.
  • the sample will include cells, tissue, or biomolecules, such as proteins, polynucleotides (e.g., DNA or RNA), organic material, and any combination of the foregoing.
  • samples include, but are not limited to, hair, skin, tissue, cultured cells, cultured cell media, and body fluids.
  • a tissue is a mass of connected cells and/or extracellular matrix material, e.g., CNS tissue, neural tissue, eye tissue, liver tissue, placental tissue, mammary gland tissue, gastrointestinal tissue, musculoskeletal tissue, genitourinary tissue, and the like, derived from, for example, a human or other mammal and includes the connecting material and the liquid material in association with the cells and/or tissues.
  • a body fluid is a liquid material derived from, for example, a human or other mammal.
  • Such body fluids include, but are not limited to, blood, plasma, serum, serum derivatives, bile, phlegm, saliva, sweat, amniotic fluid, mammary fluid, and cerebrospinal fluid (CSF), such as lumbar or ventricular CSF.
  • CSF cerebrospinal fluid
  • a sample also may be media containing cells or biological material.
  • Systems of the invention can also be used to interrogate cell lines.
  • Cell lines refer to specific cells that can grow indefinitely given the appropriate medium and conditions.
  • Systems of the invention can be used to interrogate any type of cell line.
  • Cell lines can be mammalian cell lines, insect cell lines or plant cell lines.
  • Exemplary cell lines can include tumor cell lines or stem cell lines.
  • the instrument was set up using a Lumenera Vision camera and standard Thor lab parts ( FIG. 2 ).
  • the camera sits on top with a 160 mm lens tube and a standard DIN objective with an emission filter directly above the objective lens.
  • the fluorescent excitation light source sits at about 42° above the sample stage, with the center of the beam illuminating the sample area. Multiple LEDs were set up surrounding the sample stage simultaneously.
  • UV fluorescent beads LinearFlow TM , Invitrogen
  • UV fluorescent beads at 100 and 0.8% were used to test the feasibility of the instrumental setup.
  • non fluorescent beads were also tested for nonspecific signals.
  • UV, blue, green, and red fluorescent beads (LinearFlow TM , Invitrogen) at 100% were mixed and used to test the feasibility of the multi-emission instrumental setup.
  • the images obtained ( FIG. 5 ) were overlaid using Adobe ® Photoshop.
  • the background was significantly reduced for blue, green, and UV LEDs.
  • the background reduction for blue, green, and UV LEDs were approximately 2, 8, and 3 times, respectively.
  • the detection limit was significantly improved (allowing lower fluorescence for various excitation sources or fluorophores).
  • the background reductions for blue, green, and UV LEDs were 2, 8, and 3 times respectively.
  • the signal enhancement relates to the number of LEDs linearly; therefore, where 4 LEDs are used, there are four times of enhancement.

Claims (7)

  1. System (200) zum Zählen von Zellen oder Biomolekülen, umfassend:
    eine abgedeckte Probenkammer (230) mit einer festen Tiefe im Bereich von 1 µm bis 200 µm, wobei die abgedeckte Probenkammer (230) so konfiguriert ist, dass sie ein Probenvolumen von etwa 1 µL bis 1.000 µL einer Suspension von Zellen oder Biomolekülen in einer Probe (250) aufnehmen kann, wobei die abgedeckte Probenkammer (230) ein optisch klares Fenster umfasst, das die Belichtung der Probe (250) ermöglicht;
    eine Hellfeld-Lichtquelle (220), die in der Lage ist, die Probe mit einem HellfeldLichtstrahl zu bestrahlen;
    zwei oder mehr Fluoreszenzlichtquellen (210), von denen jede in der Lage ist, unabhängig und gleichzeitig einen Fluoreszenzanregungslichtstrahl durch das Fenster auf die Probe (250) zu richten, wobei jeder Fluoreszenzanregungslichtstrahl in einem schrägen Winkel zu dem Hellfeldlichtstrahl und in einem Einfallswinkel von 10° bis 80° zur Oberflächenebene des Fensters steht;
    mindestens eine Lichterfassungsvorrichtung (240) zum Erfassen eines Lichtsignals von der Probe (250); und
    eine bewegliche Lichtblende (228) zum Steuern des Durchgangs des Hellfeld-Lichtstrahls zu der Probe (250), und wobei die Fluoreszenzlichtquellen (210) so konfiguriert sind, dass sie Licht im Bereich von 300 nm bis 10.000 nm emittieren.
  2. System (200) nach Anspruch 1, wobei das System (200) vier Fluoreszenzlichtquellen (210) umfasst, die in der Lage sind, gleichzeitig zwei Anregungslichtstrahlen mit der gleichen oder einer anderen Wellenlänge an die Probe (250) zu liefern.
  3. System (200) nach Anspruch 2, bei dem jeder der vier Fluoreszenzanregungslichtstrahl einen Einfallswinkel von etwa 45° in Bezug auf die ebene Oberfläche des Fensters hat oder denselben Einfallswinkel aufweist.
  4. System (200) nach Anspruch 1, wobei die Zellen ausgewählt sind aus der Gruppe bestehend aus Objekten, die ausgewählt sind aus Bakterien, Säugetierzellen, Insektenzellen, Pflanzenzellen.
  5. System (200) nach Anspruch 1, wobei die Zellen oder Biomoleküle ausgewählt sind aus Bakterien, Algen, Pilzen, Säugetierzellen, Insektenzellen, Pflanzenzellen, Proteinen, DNA-Molekülen und Oberflächenmarkern.
  6. System (200) nach Anspruch 1, wobei die bewegliche Lichtblende (228) so beschaffen ist, sich mechanisch
    aus der Linie mit der Hellfeld-Lichtquelle (220) herauszubewegen, damit das Licht von der Hellfeld-Lichtquelle (220) mit der Probe (250) in der abgedeckten Probenkammer (230) während der Hellfeld-Abbildung in Wechselwirkung treten kann, und
    zu bewegen in einer Linie mit der Hellfeld-Lichtquelle (220), um das Licht von der Hellfeld-Lichtquelle (220) daran zu hindern, mit der Probe (250) in der abgedeckten Probenkammer (230) während der Fluoreszenzabbildung in Wechselwirkung zu treten.
  7. System (200) nach Anspruch 1, wobei die Fluoreszenzlichtquellen (210) so konfiguriert sind, dass sie blaues, grünes oder UV-Licht emittieren.
EP11733248.6A 2010-01-12 2011-01-11 Systeme zum zählen von zellen und biomolekülen Active EP2524221B1 (de)

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EP2524221A2 (de) 2012-11-21
WO2011088014A3 (en) 2011-12-15
CN102834718A (zh) 2012-12-19
US20130099120A1 (en) 2013-04-25
EP2524221A4 (de) 2017-11-22
WO2011088014A2 (en) 2011-07-21
DK2524221T3 (da) 2022-05-23
PL2524221T3 (pl) 2022-06-20
US9329130B2 (en) 2016-05-03

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